1. Field of the Invention
The present invention relates generally to the detection and identification of bacteria in biological samples. More particularly, the present invention relates to the screening of biological samples with bacteriophage capable of specifically infecting cells of interest and transforming the infected cells to a detectable phenotype.
The detection and identification of bacterial species and strains is of interest under a variety of circumstances. For example, there is a need to be able to rapidly screen food, water, and other comestibles for contamination with pathogenic bacteria. The detection of bacteria in patient samples is similarly necessary in the treatment of numerous infectious diseases. In the latter case, it is frequently desirable to be able to specifically type the bacteria and would be further desirable to screen the bacteria for sensitivity to various bacteriocides.
Heretofore, various techniques have been used for bacterial identification, including serotyping, nutritional screening, and phage typing. Serotyping utilizes a panel of antibodies capable of binding to distinct cell surface antigens on target bacteria. Based on the observed pattern of binding, the species and strain of the bacteria may be determined. Nutritional screening relies on variations in the metabolic requirements of different types of bacteria. By growing (or attempting to grow) the bacteria on well-defined media, the bacteria may be classified based on those substances which are necessary for growth and those substances which inhibit growth.
Of particular interest to the present invention, bacteriophage have been used to type bacterial cultures based on the limited host range of different phage. By attempting to infect aliquots of a pure culture of unknown bacteria with a panel of different phage, the bacterial cell type can be determined.
While such phage typing is a highly accurate and determinative procedure for identifying bacterial type, it suffers from being both time consuming and labor intensive. Bacteria in a sample must first be grown out so that pure cultures may be isolated. Individual colonies of the pure cultures must then be grown and subsequently divided into aliquots which are exposed to the different phage in the panel. After exposure, conventional plaque assays are run to determine the infectivity of the various phage. The entire procedure takes from 24 to 48 hours, or longer, and requires highly trained personnel for execution. Because of the lengthy procedure, and the need to identify plaques in the bacterial colonies, the procedure is not amenable to automation. Moreover, because of the time required, the procedure is less than ideal for determining the nature of a patient's infection prior to therapy.
Disabled bacteria, such as those debilitated by cooking or partial heat sterilization, are a major detection problem in many food processing situations. Such disabled bacteria frequently remain viable (and thus potentially pathogenic) yet are sufficiently weakened so that detection by conventional assay protocols may require a non-selective recovery step (pre-enrichment) followed by a selective enrichment step to allow growth of the targeted bacteria while growth of competing organisms is inhibited. Such additional steps can significantly add to the time required to perform the assay.
Detection of particular bacteria in open environments, such as air, water, and soil, can also be problematic. Because of the wide variety of species that may be present, it will often be difficult to distinguish a particular species of interest.
In view of the above, it would be desirable to provide improved phage screening assays for detecting and identifying bacterial cells in biological samples. In particular, it would be desirable if such assays could be performed in a relatively short period of time and that the assay protocols be sufficiently simple to be performed by semi-skilled personnel. Preferably, the assays will be performed on mixed cultures, with a minimum number of steps, and result in a detectable event which is easily observed and amenable to automated reading. It would be further desirable if the assays were able to detect partially disabled bacteria which might otherwise require a pre-enrichment step and a selective enrichment step for detection. In addition, it would be desirable to be able to rapidly and conveniently detect particular bacterial cells in open environments, such as air, water, and soil.
2. Description of the Background Art
The use of bacteriophage in characterizing the surface of bacterial cells is discussed in Makela, Enterobacterial Surface Antigens: Methods for Molecular Characterization, Korhonen et al., eds., Elsevier Science Publishing, Amsterdam, pp. 155-178 (1985), where conventional plaque assays are employed to determine infectivity of particular phage. The molecular genetics of bacteriophage P22 is discussed in Susskind and Botstein (1978) Microbiol. Rev. 42:385-413. The ability of P22 to act as a transduction vector is described in Watanabe et al. (1972) Virol. 50:874-882 and Orbach and Jackson (1982) J. Bacteriol. 149:985-994. The use of P22 to selectively transduce recombinant plasmids with integrated pac sequences is described in Schmidt and Schmieger (1984) Mol. Gen. Genet. 196:123-128 and Vogel and Schmieger (1986) Mol. Gen. Genet. 205:563-567. Foreign genes are inserted into bacteriophage L (related to P22) by transposon mutagenesis, as described in Spanova and Karlovsky (1986) Folia Microbiol. 31:353-363. The use of .lambda. phage as a cloning vector is described in Young and Davis (1983) Proc. Natl. Acad. Sci. USA 80:1194-1198, where unknown gene products may be detected by antibody probes. The use of M13 phage as a cloning vector is described in Vieira and Messing (1987) Meths. Enz. 153:3-11.
Bacteria may be detected in biological samples by a number of techniques, including selective media, immunoassays, and nucleic acid probes. Particular methods for detecting Salmonella are described in U.S. Pat. No. 4,689,295. A phage-based test for detecting Salmonella in food is described in ASM News (1987) 53:542. The test uses phage to mediate the adsorption of the target Salmonella on a surface.
The ability to nucleate ice formation has been reported to be encoded by a single gene in several ice nucleation-positive (Ina+) bacteria, and this ability can be transferred to E. coli by transformation with a plasmid carrying the ice nucleation gene. See, U.S. Pat. No. 4,464,473; Orser et al., Molecular Genetics of the Bacterial-Plant Interaction (A. Puhler, ed.), Elsevier/North Holland Biomedical, pp. 353-361 (1983); Green et al. (1985) Nature 317:645-648; and Corotto et al. (1986) EMBO J. 5:231-236. Sequence information for an ice nucleation gene in P. syringae (gene inaZ) has been reported; Green et al. (1985) id. The corresponding protein is of approximate molecular weight 1.2.times.10.sup.5. Sequence information for an ice nucleation gene in P. fluorescens (gene inaW) has also been reported. See, Warren et al. (1986) Nuc. Acids. Res. 14:8047-8060. Information concerning the identification and purification of the inaZ and inaW proteins is reported in Wolber et al. (1986) Proc. Natl. Acad. Sci. USA 83:7256-7260.
The droplet freezing assay is a known method of testing for the presence of whole cell ice nucleating bacteria and cell-free nuclei. The method consists of laying out an array of N droplets of volume V (usually 0.01 ml) on a nucleus-free surface, cooling to temperature T (less than 0.degree. C.) and scoring N.sub.f, the number of droplets frozen. The number of nuclei/ml is then calculated by the following formula: nuclei/ml=(1/V) log.sub.e [N/(N-N.sub.f)]. G. Vali (1971) J. Atmos. Sci. 28:402-409.